Food Quality Safety

148 L.F. Fleuri et al. such as insects, nematodes, and fungi. Proteases catalyze the hydrolysis of peptide bonds of proteins and may have activity on ester and amide bonds. Lipases allow catalysis of the hydrolysis and synthesis, often in chemo, regal, or enantioselective reactions. Furthermore, phytase catalyzes the hydrolysis of phytate to phosphate and inorganic phosphorus, increasing the bioavailability of phosphorus for mono- gastric animals. Keywords Enzymes • Solid state fermentation • Bioprospecting • Fungus • Actinomycetes • Biomolecules 10.1 Introduction The exploitation of biodiversity rises as a new exploitation method of biological natural resources, generating bioprospecting, that is defined as the method to determinate, evaluate, and explore legally and systematic life diversity in particular location, whose main goal is seek for genetic and biochemical resources for commercial purposes. Therefore, microorganisms are versatile for molecules pro- duction with biological activities by fermentation processes, such as the solid state fermentation (SSF). The SSF is widely used for obtaining biotechnological products, and it has become an interesting alternative to reduce the processes cost. This type of fermen- tation can use agricultural and agro industrial waste as substrates, which present low value, are nutrient rich, and have restricted water availability that helps to select contaminants, especially bacteria and yeasts. The obtainment of the final product by SSF is easier and the amount of waste is minimized (Lima et al. 2003). The use of surplus/waste as substrate for SSF allows the reduction of the final product cost and the implementation of a closed, sustainable, and environmentally friendly product chain. Among these substrates we can mention sawdust, bagasse from sugarcane of the sugar and alcohol industry and straw, bark and bran from cereal and fruit production. For biotechnological processes, the microorganisms are widely required because they are, in most cases, unicellular; when they are multi- cellular, they are poorly differentiated; they simplify cultivation in fermenter; they have rapid absorption of nutrients, fast metabolism, and high versatility, transforming different compounds and producing a wide variety of products. Microorganism is considered viable for a process when it is able to grow on cheap substrates; it is genetically stable, but liable for genetic manipulation; it provides high production yields on large scale, and, also, recovered at low cost; it does not produce incompatible substances with the target final product and it is not pathogenic. Actinomyces and fungi are the most used by SSF, since they grow under low water activity conditions. Actinomyces were originally classified as fungi, as they present aerial hyphae, however, detailed studies of the cell wall composition, particularly the lipid mem- brane and the composition of its peptidoglycan, classified them as true aerobic bacteria. Molecular taxonomy studies created the class Actinobacteria, which includes all gram-positive bacteria with guanine and cytosine content greater than

10 Exploration of Microorganisms Producing Bioactive Molecules of Industrial. . . 149 55 %. Within this new class, actinomyces with capacity to produce mycelium are classified as Actinomycetales and include 10 subclasses and 34 families. Each year, new proposals are presented in literature of new species, genera, or families, and so, the classification of these organisms is constantly renewed (Stackebrandt et al. 1997; Stackebrandt 2000). Actinomyces of genus Streptomyces, the most commonly isolated and studied, are considered important microorganisms for industrial production and they have been described as the main antibiotics producers. Species of this genus are noted for producing more than half of the 10,000 bioactive compounds documented until 2001 (Anderson and Wellington 2001). Due to its high metabolic diversity, actinomyces have also been explored as major producers of many bioactive substances (Korn-Wendisch and Scheider 1992). The Kingdom Fungi consists of about 1.5 million species of which 77,000 species are known. These microorganisms have important ecological functions in nature, such as decomposition of organic material and reduction of mineral dis- charge to environment, immobilization and nutrient release, association with plants that can vary from beneficial to pathogenic, release of organic acids for the soil, among others. They are capable of degrading various substances with aid of exoenzymes to achieve required solubility and shape to be transported and incorporated by the cells. These enzymes are amylases, pectinases, xylanases, lipases, cellulases, and proteases, which are important for many applications in promising industrial processes (Silva et al. 2008). Furthermore, they produce other metabolites as antibiotics, chelating agents, and others (Hawksworth et al. 1996; Fransson et al. 2004; Klein and Paschke 2004). 10.2 Production of Bioactive Substances by Microorganisms The production of bioactive compounds by SSF may be conducted as shown in Fig. 10.1. Actinomyces are producers of antibiotics (Bull 2004; Berdy 2005; Strohl 2004), antitumor agents (Olano et al. 2009), and immunosuppressive agents (Mann 2001). The Streptomyces have the ability to produce many bioactive compounds. Around 23,000 antibiotics have been discovered from microorganisms. It is estimated that about 10,000 of them have been isolated from actinomyces (Okami and Hotta 1988). Regardless of its chemical structure, these bioactive substances can be classified as peptides, quinones, macrolides, terpenes, polyketides, among others (Li and Piel 2002; Salmon et al. 2003). Most peptides derived from Streptomyces species are cyclical and contain elements such as chromophores or amino acids in its structure. Peptides include ciclomarine A, which can be obtained from Streptomyces with great anti-inflammatory and antiviral activity (Renner et al. 1999), and piperazimicines A-C, which are cytotoxins isolated from Streptomyces

150 L.F. Fleuri et al. Fig. 10.1 Solid state fermentation process. Microorganisms kept in slants are sterile inoculated in reactors containing substrate previously sterilized. Afterwards they are incubated for fermentation, followed by water addition, homogenization and filtration to obtain the substances with biological activity sp. Though, piperazimicine A showed high cytotoxicity against tumor cells in vitro (Miller et al. 2007) and salinamides A and B, produced by Streptomyces sp. CNB-091, showed anti-inflammatory activity (Moore et al. 1999). Quinones are compounds with conjugated dione cyclic in its structure and they are common constituents and biologically relevant molecules. As an example: C-glycoside himalomicines A and B complex and tetracenomycin D. The first is the anthraqui- none with fridamicine E chromophore, precursor of anthracycline antibiotic obtained from Streptomyces sp. 6921, that show great antibacterial activity (Maskey et al. 2004), while the second, the anthraquinone antibiotic produced by Streptomyces corchorusii AUBN (Adinarayana et al. 2006) showed cytotoxic activity against hepatic carcinoma cells. Terpenes (large and diverse class of hydrocarbons) are biosynthetically derived from units of isoprene, with molecular formula C5H8. Streptomyces sp. NPS008187, isolated in Alaska, synthesized three

10 Exploration of Microorganisms Producing Bioactive Molecules of Industrial. . . 151 new pyroles sesquiterpenes which showed antibacterial activity (Macherla et al. 2005). Carotenoids are tetraterpenos mostly know, and they can be obtained from strains such as Streptomyces griseus (Lee et al. 2001). The polysaccharides produced by basidiomycetes fungi are extensively studied in China and Japan, due to its medicinal and tonics attributes. Examples include Agaricus blazei which produces substances with anticarcinogenic activity (Mizuno et al. 1990); Flammulina velutipes that produces elements which helps to reduce cholesterol (Miles and Chang 1997); extracts of Ganoderma lucidum which are immune system boosters and promoters of tonic effects for cardiac system (Hikino et al. 1985); Lentinula edodes biomolecules with anti-HIV effect (Chihara 1992); and others. Filamentous fungi (ascomycetes) of Penicillium genus are fairly flexible for antibiotics production by fermentation processes alone or associated with chemical modification, such as penicillin G produced by P. chrysogenum; griseo- fulvin of P. griseofulvin used for infections treatment of skin, hair, and nails; cyclosporin, used as an immunosuppressant in transplant surgery; and fusidic acid, used to help control the infection by Staphylococcus aureus resistant to methicillin. Duarte et al. (2012) described the achievement of marine fungi molecules from different genus such as Penicillium, Fusarium, Trichoderma, Hupocrea, Phoma, and Scopulariopsis, among others with cytotoxic, antifungal anticoccidial, antiviral, and neuroactive activity. Whereas Xiong et al. (2009) stud- ied the production of antibacterial compounds by Cladosporium sp., Meenupriya and Thangaraj (2011) describe the bioactive molecules obtained from marine organisms present anticancer, antimicrobial, and anti-inflammatory activity. Thus, these researchers obtained molecules from Aspergillus ochraceus with activity against microorganisms that cause humans diseases. 10.3 Production of Enzymes by Microorganismis Enzymes include a abundant class of substances produced by actinomyces and fungi. The advantages of using microorganisms for enzyme production in replace- ment of the traditional animal and vegetable sources are relatively high perfor- mance, low cost, and susceptibility to genetic manipulation. Currently, microorganism enzymes are used in food processing, manufacture of detergents, textile and pharmaceutical industries, medical therapy, molecular biology, biofuels industry, wastewater treatment, environmental preservation, bioremediation, and biological control. These microorganisms have a wide ecological and biochemical diversity, and furthermore, they have a high capacity for production of secondary metabolites. Therefore, they can be considered an excellent source for finding new enzymes with new specificities and different biochemical characteristics. They are capable of producing several enzymes that can be considered promising for bio- technological applications, including oxidoreductases, transferases, hydrolases, lyases, isomerases, and synthases. Hydrolases are noteworthy, because these are cellulases, hemicellulases, proteases, chitinases, phytases, and lipases, whose features and applications are described below.

152 L.F. Fleuri et al. 10.3.1 Cellulases Cellulases are enzymes consisting of complex capable of acting on cellulosic materials, promoting its hydrolysis. These biocatalysts enzymes are highly specific, acting synergistically to release sugars, where glucose is the one with greater industrial interest due to the possibility of its conversion to ethanol, sweeteners, phytohormones, organic acids, etc. The steps involved in cellulose degradation by cellulase are not fully understood, but it is formed as a multienzyme system including three enzymes that act together for hydrolysis of cellulose: endoglucanases (EC 3.2.1.4), which cleave randomly cellulose polymer by chang- ing the degree of polymerization; cellobiohydrolases (EC 3.2.1.91), which hydro- lyze the polymer at its nonreducing end, releasing cellobiose; and cellobiases (β-glucosidase, EC 3.2.1.21), which are responsible for cleavage of small chain, both celloligosaccharides and cellobiose, until glucose (Fleuri and Lima 2013). The prospect of cellulose degradation (most abundant polymer in nature present in vegetable cells) is linked to program implemented in Brazil in 1970 that meant to replace gasoline with ethanol from sugarcane. Consequently, research for agricul- ture and new technologies have been greatly intensified, leading Brazil in a favorable position in terms of secure energy sources. However, only a part of the biomass produced is used for bioenergy production, as one-third of the sugarcane is used for sugar production, one-third is residue, which is burned to produce electric- ity, and the other third of the remaining residue is left in the field and decomposed by microorganisms (Zanin et al. 2000; Soccol et al. 2010). However, a significant increase in ethanol production may be possible if new technologies converting the polysaccharides of the two-thirds of the remaining biomass of the entire process in bioethanol. For the last four decades, much effort is being made to development of second-generation bioethanol, through abundant and renewable lignocellulose bio- mass by physical, chemical, and enzymatic treatments, isolation and/or combined (Hahn-Ha¨gerdal et al. 2006; D’Souza-Ticlo et al. 2010; Soni et al. 2010). The raw lignocellulose materials include agribusiness, municipal waste, and wood from angiosperms and gymnosperms. The agro industrial materials are important for its residue character, after processing raw materials with high value, and the natural capacity that Brazil has for generation of these products, that is: sugarcane bagasse and straw, soybean straw, rice straw, and corncobs. Among the mentioned biomass, bagasse from sugarcane is predominant in Brazil, producing, in 2007, 147 million tons of wet mass (Chandra et al. 2010). Furthermore, these materials may also be used for solid state fermentation (SSF), since they are inexpensive materials and they have shown effective results for biocatalysts and bioactive compound produc- tion (Lever et al. 2010; Sukumaran et al. 2009; Bhattacharya and Banerjee 2008; Lin and Tanaka 2006; Mishima et al. 2006). The polysaccharides present in lignocellulose biomass must be hydrolyzed with acid (in the presence of high temperature and pressure) and/or cellulases and other enzymes to release ferment- able sugar in a high yield. Pre-treatment help to hydrolyse the lignin and to solubilize the cellulose partly, so the enzyme can act on the molecule and available

10 Exploration of Microorganisms Producing Bioactive Molecules of Industrial. . . 153 all the remaining hexoses and pentoses. The process of enzymatic conversion of lignocellulose into ethanol is affected by the use and purchase of cellulases prepa- ration, since they are marketed by a small number of suppliers and have high cost. For this process to become economically viable, large-scale production of cellulases at low cost, using agro-industrial residues as substrate, is necessary (Maclean and Spatari 2009; Chandra et al. 2009). Actinomyces produce cellulase with high activity and stability in extreme temperature and pH conditions (Lima et al. 2005; Jang and Chen 2003). Such cellulases exhibit great activity in a wide range of pH, between 4.0 and 8.0, which it is also promising (Lima et al. 2005; Jang and Chen 2003; George et al. 2001; Bhat 2000). The proportion of current total production of cellulases as additives for detergents for laundry industry market exceeds 30 %. Due the increase of environmental pressure on paper and textile industries, it is assumed that cellulases should play an important role in the development of clean technology, both for denim processing and for discoloration of paper for recycling purposes (George et al. 2001). Currently, one of the main applications for cellulases are textile industry, where the need of high temperatures (50–65 C) and alkaline pH requires the use of thermostable enzymes for efficient jeans treatment (Bhat 2000). The main commercial cellulase preparations are obtained from filamentous fungi, such as Aspergillus niger (Cellulocast of Novozyme) and Trichoderma reesei (Megazyme). Among cellulases producing fungi, we can name genus Aspergillus, Trichoderma, Penicillium pinophilum, Sporotrichum, Fusarium, Talaromyces, Thermoascus, Chaetomium, Humicola, Neocallimastix, Piromonas, and Sphaeromonas (Fleuri and Lima 2013). 10.3.2 Xylanases Xylanases enzymes act on xylan, hemicellulose components, which may be associated to cellulose and lignin in the plant cell wall. Xylan is formed by xylose units linked with β-1,4 glycosidic bonds; they, also, may have arabinose, glucuronic acid or 4-methyl ether, and acetic, p-cumaric, and ferulic acids (Brienzo et al. 2008). Among the xylanases enzymes, there are β-1,4 endoxylanases (β-1,4-D-xilanil-xylan hydrolase, EC 3.2.1.8), which depolymerize xylan by ran- dom hydrolysis of main skeleton, and β-xylosidases (β-1,4-D-xilosidic-xylo hydro- lase, EC 3.2.1.37), which hydrolyze small oligosaccharides (Collins et al. 2005). Xylanase’s most important application is in the pulp and paper industry where high temperatures (55–70 C) and alkaline pH of the pulp substrate requires utilization of thermostable enzymes for efficient bleaching (Beg et al. 2001; Collins et al. 2005; Saha 2003). However, other applications, such as food industry can be mentioned like: dough preparation (Collins et al. 2005), for clarification of beer and juices, and partial hydrolysis of xylan in animal feeds. Nascimento et al. (2003) found that the xylanase extract obtained from Streptomyces malaysiensis showed biochemical characteristics (temperature 50–65 C and pH 6.0–8.0) with great potential for

154 L.F. Fleuri et al. pulp and paper industry. Beg et al. (2000) showed optimal values of temperature, range between 50 and 75 C and pH from 6.0 to 9.0 for strain Streptomyces QG-11- 3. Most known thermostable xylanases are produced by strains of Thermatoga, with half-life of 90 min at 95 C (Sunna and Antranikian 1997). However, very signifi- cant thermostability of xylanases has been studied in many Streptomyces strains, including Streptomyces sp. T7 with stability at 50 C, at pH 6.0 for 6 days (Deng et al. 2005). Costa et al. (2000) described the production of xylanase complex using Penicillium janthinellum with sugarcane bagasse hydrolyzed as substrate. 10.3.3 Proteases Proteases (EC 3.4.21.12) catalyze hydrolysis of peptide bonds of proteins, and they may have activity on ester and amide bonds. The proteolytic enzymes synthesized by microorganisms have become significant for research because of its wide application at different industries and medicine, as well as its involvement in microbial metabolism. They are used in leather industry, pharmaceutical and food industries, in hydrolysis of substrates used for microbiological growth and paren- teral nutrition preparation, detergents, and cosmetics. Proteases enzyme preparations are particularly important in medicine for burns cleaning and removal of necrotic tissue and blood clots lysis (Landau and Egorov 1996). Proteases can also be applied for monogastric animals feed aimed at reduction of anti-nutritional agents of vegetable ingredients, increased digestibility, increasing endogenous enzymes activity, and reduction of environmental pollution (Garc´ıa et al. 2000). Actinomyces and fungi produce a variety of extracellular peptidases, including endopeptidases (serine and metallo-peptidases, specially) and exopeptidases (amino- and carboxypeptidases) with specificity for many substrates. Peptidases obtained from actinomyces, such as serine-peptidases from Streptomyces exfoliates; serine and metallo-peptidases from Streptomyces lactamdurans; and serine-peptidase from Streptomyces pactum are involved in the nitrogen protein sources assimilation, in degradation of aerial mycelium, in sporulation processes, and in production of antibiotics (Kim and Lee 1996). Peptidases and other enzymes used in detergent formulations may have high activity and stability in a wide range of pH and temperature. Serine and metallo-peptidases have been described for the genus Streptomyces, as observed with strains Streptomyces sp. 594, Streptomyces malaysiensis AMT-3, and Streptomyces alboniger (Born 1952). However, the nature and characteristics of each component of the peptidase complex from Streptomyces has not been extensively studied. Likewise, thermostable actinomy- ces produce peptidase with major role in degradation of keratin components, such as chicken feathers present in the poultry industry waste (De Azeredo et al. 2004). Specifically, keratinase produced by actinomyces can have great biochemical characteristics with pH ranging between 6.0 and 9.0 and optimal temperature between 50 and 70 C, as observed for some species (Gushterova et al. 2005). Fungi are, also, capable for protease production. Zanphorlin et al. (2011) used

10 Exploration of Microorganisms Producing Bioactive Molecules of Industrial. . . 155 wheat bran moistened with casein and nutrient for protease production using fungus Myceliophthora sp. The enzyme showed optimum pH and temperature of 9.0 and 40–45 C, respectively. Rojas et al. (2009) studied fungal proteases obtained from Eladia sacculum in biodeterioration processes. Cabaleiro et al. (2002) studied protease production by fungi Phanerochaete chrysosporium and Phlebia radiata in SSF using nylon sponge and corncob. Proteases obtained from this process were distinguished by microbial growth time and activity, and they are of different classes. 10.3.4 Chitinases Chitin is linear polymer of β-1,4-N-acetylglucosamine, which is the most abundant natural amino polysaccharide. Moreover, it is present in cell wall of most fungi and it is the main constituent of insects and crustaceans exoskeleton (Fleuri et al. 2009a). The hydrolysis of chitin occurs by action of enzyme complex involving two enzymes: chitinase or poly (1,4-N-acetyl-β-D-glicosaminida) glucan hydrolase (EC 3.2.14), which breaks randomly internal bonds in the chitin chain, generating oligomers and disaccharides, and β-N-acetyl-glucosaminidase or β-N- acetyl-β-D-hexosaminide-N-acetyl-hexosamino hydrolase (EC 3.2.1.52), which cleaves nonreducing terminal unit, releasing N-acetylglucosamine. The first ones have higher affinity for larger molecules, while the others prefer small oligomers, including quitobiose (Merzendorfer and Zimoch 2003). Study of chitinase has been increasing because its contribution as defense agents against pathogenic organisms that have chitin, such as insects, nematodes, and fungi (Sahai and Manocha 1993). Besides, chitinases can be used as a protective agent against pathogenic fungi, in protoplast preparation, and production of biologically active substances as aminoglucanooligossacarides (Fleuri et al. 2009a, b). Han et al. (2008) observed application of chitinase in medicine (hypocholesterolemic action and antihyperten- sive), in agriculture (anti-phytopathogenic), in bioremediation, and in maintenance of food quality. It is estimated that there are between 10 and 25 different chitinases. Tikhonov et al. (1998) produced and purified chitinases from Streptomyces kurssanovii. Brzezinska et al. (2012) studied the degradation of chitin substances with chitinase from Streptomyces rimosus, which was isolated from soil. Many fungi genus (Beauveria sp., Aspergillus sp., Thermoascus sp., Chaetomium sp. Trichoderma sp.) are able to produce chitinases by SSF. 10.3.5 Lipases Lipases are enzymes that are increasing at the biotechnological enzymes scenario. They are very versatile, allow catalysis of hydrolysis and synthesis of chemical reactions; often in chemo, regal, or enantioselective, lipases are applied in many industries such as food industry, pharmaceuticals, fine chemicals, oil chemistry,

156 L.F. Fleuri et al. detergents, and biodiesel (Barros et al. 2010). The participation of lipases in the world market of industrial enzymes has grown significantly; it is estimated that in the future they will have world significance comparable to peptidases today which count for 25–40 % of industrial enzymes sales (Hasan et al. 2006). Lipases act in the organic–aqueous interface; they catalyze hydrolysis of carboxylic-ester bonds and liberate fatty acids and organic alcohols. However, the reverse reaction (esterifica- tion) and also various transesterification reactions can occur in environments with restricted water (Freire and Castilho 2008). The transesterification term refers to radical change between an ester and an acid (acidolysis), or ester and alcohol (alcoholysis), or between two esters (interesterification). Their ability to catalyze these reactions with high efficiency, stability, and versatility make these enzymes very commercially important. Lipases are enzymes of the group of serine hydrolases (EC 3.1.1.3). Their natural substrates are triglycerides; however, its activity is increased when located at the interface polar/nonpolar, and they have higher affinity for long-chain fatty acids (Hasan et al. 2006). Among lipases obtained from actinomyces, there is Streptomyces rimosus, S. coelicolor (Co¨te and Shareck 2008), S. fradiae, S. coelicolor (Sharma et al. 2001), S. exfoliatus, S. albus, and S. cinnamomeus (Abramic et al. 1999). Bielen et al. (2009) and Abramic et al. (1999) reported that lipases have been traced from different microorganisms for different kinds of applications, and that streptomycetes have a large number of genes encoding different enzymes with many lipolysis activities. Among these actinomyces are cited S. exfoliates, S. albus, S. coelicolor, S. rimosus, and S. exfoliatus. Mander et al. (2012) studied the transesterification with the lipase obtained from Streptomyces sp. CS133 for production of biodiesel. Even with a wide variety of microbial lipases, use of these enzymes on industrial scale is still limited due to high production costs, low activity, and limited biochemical characteristics, which facilitates searching of other microbial lipases sources. Extracellular lipases from fungi Rhizopus homothallicus (thermostable) were obtained by SSF with sugarcane bagasse as substrate. Moreover, these authors mention that the yield of enzyme production by SSF is higher than liquid fermenta- tion due to increased rate of biomass growth. There are lower protease production that can degrade other enzymes, as well as higher stability for pH and temperature of the enzyme obtained by this type of fermentation (Mateos Diaz et al. 2006). The main commercial lipase preparation is from Aspergillus oryzae, created from lipase clones derived from Thermomyces lanuginosa (Lipolase from Novo Nordisk) and lipase clones from Rhizomucor miehei (Lipozyme, Novo Nordisk S/A). They are especially applied as detergents and production of analogues of cocoa butter from cheap oil sources (Romdhane et al. 2010). 10.3.6 Phytases Phosphorus is an important ingredient for various biochemical pathways, biological processes, and skeletal integrity. Vegetable ingredients are important sources of

10 Exploration of Microorganisms Producing Bioactive Molecules of Industrial. . . 157 phosphorus, and phytate (inositol hexaphosphate or IP6) is the mineral storage for plants. The amount can differ between plant species. However, phytic acid is not a suitable source of phosphorus for nonruminants animals, since 85 % of the phos- phorus is bound to inositol making phytic acid or inositol hexametaphosphate, kept it chelated and unavailable. Diets fed to animals are supplemented with inorganic sources of phosphorus such as calcium phosphate or animal sources like meat and bone flour, due to the lack of availability of phosphorus and a possible deficiency of this mineral for animals in diets with vegetable ingredients. As result, diets for nonruminant animals have amount of phosphorus addition to nutritional requirements, with elimination of excess not absorbed by the animal. Furthermore, phytate acts as anti-nutrient associated to proteins, amino acids, lipids, and minerals, while it interacts with their digestive enzymes reducing activity, influencing digestion, and impairing nutrients utilization. In this sense, phytase catalyzes the hydrolysis of phosphate and phytic acid to phosphorus inorganic, increasing the bioavailability of phosphorus for monogastric animals. Phytase classification is based on first position of the phosphate to be hydrolyzed; named 3-phytase (EC 3.1.3.8) and 6-phytase (EC 3.1.3.26). Supplementation of phytase in diet benefits animal nutrition and improves digestibility of protein, gross energy, and increases the availability of calcium, phosphorus, zinc, manganese, and mag- nesium. Furthermore, these enzymes improve phosphorus availability in 50 %, and it is important toll to reduce environmental excretion, because of better utilization of phytic phosphorus from vegetable sources, reducing utilization of inorganic sources. Main phytases are classified as their activity on determined pH. Acid phytases show better dephosphorylating between pH 5.0 and alkaline phytase in pH 8.0. All phytases show great pH between 4.0 and 6.0 (Kies et al. 2001; Lei and Stahl 2000). Phytase is produced in large commercial scale by recombination DNA techniques, from fungi of genus Aspergillus niger. Enzymes that blend with phytase from Peniophora lycii, Schizosaccharomyces pombe, and Escherichia coli are also found on the market. References Abramic M, Lescic I, Korica T et al (1999) Purification and properties of extracellular lipase from Streptomyces rimosus. Enzyme Microb Technol 25:522–529 Adinarayana G, Venkateshan MR, Bpiraju VV et al (2006) Cytotoxic compounds from the marine actinobacterium. Bioorg Khim 32:328–334 Anderson AS, Wellington EMH (2001) The taxonomy of Streptomyces and related genera. Int J Syst Bacteriol 51:797–814 Barros M, Fleuri LF, Macedo GA (2010) Seed lipases: sources, applications and properties – a review. Braz J Chem Eng 27:15–29 Beg QK, Brushan B, Kapoor M et al (2000) Production and characterization of thermostable xylanase and pectinase from Streptomyces sp. QG-11-3. J Ind Microbiol Biotechnol 24:396–402 Beg QK, Kapoor M, Mahajan L et al (2001) Microbial xylanases and their industrial applications: a review. Appl Microbiol Biotechnol 56:326–338

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Chapter 11 Recent Biosensors for Food Analysis in Brazil and Italy Valber A. Pedrosa, Luciana F. Fleuri, Giuseppina P.P. Lima, Massimiliano Magro, and Fabio Vianello Abstract The importance of safe and high-quality food products is doubtless and consumer demand for increased food quality and safety assurances moves down the chain with retailers and service providers asking suppliers and producers to provide verifiable proof that robust food quality and safety control systems have been effectively implemented. Food analysis needs rapid and reliable methods to ensure the quality of products and process control. Food quality control is essential both for consumer protection and also for food industry. Nowadays, the convergence of new technologies, including biotechnology, nanotechnology, and electronic technology, has opened new horizons in development of biosensors. These devices offer advantages as alternatives to conventional methods because they enable real-time detection, portability, and fast laboratory or in-field analysis. This contribution presents a review about the development and application of biosensor technology in foods, and future trends in Brazil and Italy. Keywords Biosensors • Food quality • Food safety • Food analysis • Food contamination V.A. Pedrosa (*) • L.F. Fleuri • G.P.P. Lima Department of Chemistry and Biochemistry, Insituto de Biocieˆncias, Campus de Botucatu, Universidade Estadual Paulista (UNESP), Sa˜o Paulo, Brazil e-mail: [email protected]; [email protected]; [email protected] M. Magro • F. Vianello Department of Comparative Biomedicine and Food Science, University of Padua, vialedell’Universita` 16, 35020 Legnaro, Padova, Italy Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Palacky University in Olomouc, Olomouc, Czech Republic e-mail: [email protected]; [email protected] G.P.P. Lima and F. Vianello (eds.), Food Quality, Safety and Technology, 163 DOI 10.1007/978-3-7091-1640-1_11, © Springer-Verlag Wien 2013

164 V.A. Pedrosa et al. 11.1 Background In recent years, food-safety emergencies have shaken consumer confidence in the food production chains, focusing attention on how food is produced, processed, and marketed. National Health Agencies, around the world, have recognized food safety and food quality as a top priority. They have established new policies, modernizing legislation into a coherent and transparent set of rules, which can guarantee high level of consumer protection and, thus, human health. An effective food safety policy requires the assessment and monitoring of the risks to consumer health, associated with the presence of contaminants in raw materials, farming practices, and food processing activities. In the course of the third Spring School organized by the Brazilian State University of Sao Paulo (UNESP) and the Italian University of Padova (Italy) in the Botucatu Campus (Bioscience Institute) in September 18–20th, 2012, these themes were faced and in this chapter recent biosensors developed in the two countries are reviewed. In fact, the rigorous control of food quality and safety is of growing interest for both consumers and food industries. In the food industry, the quality of a product is evaluated by periodic chemical and microbiological analysis. It has become impor- tant to periodically measure a variety of contaminants in food, such as bacteria, viruses, natural toxins, chemical compounds (pesticides, toxic metals, veterinary drugs residues, undesirable fermentation products), and packaging materials. Most of the toxic agents found in foods are natural contaminants from environmental sources, but some of them are chemical compounds deliberately added during food processing (Codex 2009). Consumers are concerned about long-term impacts of mixtures of these chemical additives (pesticides, toxic metals, flavorings, and colors) and about their chronic, as well as, acute effects, especially on children (Jackson 2009). While the knowledge of phytochemical effects on human health and risks from chemical residues in food has led to a growing interest and attention towards the fast growth of functional and enriched food on the market, only little emphasis has been placed on the analytical aspects (Lavecchia et al. 2013). In this regard, specific, new technologies have recently been developed to examine food components and different analytical procedures were developed to assess food quality and to determine food contaminants. Normally, these procedures are based on various instrumental techniques, such as chromatography, spectrophotometry, electropho- resis, titration etc. These analytical procedures do not easily allow continuous monitoring, mainly because they are based on expensive instrumentations and they need time-consuming multistep sample extraction and pretreatments and well-trained operators, which increase the time and cost of the analysis. Meanwhile, National Health agencies and food industry request affordable methods to deter- mine compounds of interest in foods. The demand for fast and real-time analyses aimed at the detection of substance related to food quality and safety led to rapid advancements in biosensors technol- ogy (Mozaz et al. 2004). These compact analytical devices incorporate a biological

11 Recent Biosensors for Food Analysis in Brazil and Italy 165 Fig. 11.1 Schematic drawing of a biosensor sensing element, either closely connected to or integrated within a transducer system able to convert a biological event in an electrical signal (see Fig. 11.1). The recognition mechanism is based on the interaction of biological recognition component with the analyte of interest, by different recognition mechanisms. Information, which is produced in the recognition event, is transformed by means of the transducer into a signal. The Physical Chemistry and Analytical Chemistry Divisions of IUPAC (Thevenot et al. 1999) state a definition applicable to electro- chemical biosensors: “A biosensor is a self-contained integrated device that is capable of providing specific quantitative or semi-quantitative analytical informa- tion using a biological recognition element (biochemical receptor) which is in direct spatial contact with a transduction element.” Four major types of transducers can be found: electrochemical, mass, optical, and thermal (Thevenot et al. 1999). They have been used to develop biosensor aimed at the detection of a broad spectrum of analytes present in complex sample matrices. Great promises in different areas, such as clinical diagnostics, food analysis, and environmental monitoring (Table 11.1), have been proposed and the sensitivity of a particular sensor system varies depending on the transducer’s properties and the biological recognizing element. In general, biosensors consist of three main components as shown in Fig. 11.1: a recognition element, a transducer unit, and a controlling electronic unit, including an input/output interface. The recognition element, which binds to the analyte of interest, is the component producing the primary signal. The transducer represents the biosensor component, which is responsible for the transformation of the primary signal, coming from the recognition element, to a form that can be filtered, amplified, and transferred to the electronic component, which finally processes and displays, and even stores, the analytical result. The choice of the biological

166 V.A. Pedrosa et al. Table 11.1 Most common transducers used in biosensor development Transducer Advantages Disadvantages Application Ion-selective Simple, reliable, easy to Sluggish response, Amino acids, electrode transport requires a stable ref- carbohydrates, erence electrode, sus- alcohols, and inorganic ceptible to electronic ions noise Electrodes Simple, extensive variety Low sensitivity, multiple Glucose, galactose, lac- of redox reaction for membranes, or tate, sucrose, aspar- construction of the enzyme can be neces- tame, acetic acid, biosensors, facility for sary for selectivity and glycerides, biological miniaturize adequate sensitivity oxygen demand, cadaverine, histamine, etc. FET Low cost, mass produc- Temperature sensitive, Carbohydrates, carboxylic tion, stable output, fabrication of different acids, alcohols, and requires very small layer on the gate has herbicide amount of biological not been perfected material, monitors several analytes simultaneously Optical Remote sensing, low cost, Interference from ambient Carbohydrates, alcohols, miniaturizable, multi- light, requires high- pesticide, monitoring ple modes: absor- energy sources, only process, bacteria, and bance, reflectance, applicable to a narrow others fluorescence, extensive concentration range, electromagnetic range miniaturization con can be used affect the magnitude of the signal Thermal Versatility, free from No selectivity with the Carbohydrates, sucrose, optical interferences exception of when alcohols, lipids, such as color and used in arrangement amines turbidity Piezoelectric Fast response, simple, sta- Low sensitivity in liquid, Carbohydrates, patho- ble, output, low-cost, interference due to genic microorganisms, or readout device, no nonspecific binding contaminants (e.g., special sample antibiotics, fungicides, handling, good for gas pesticides), toxic rec- analysis ognition as bacterial toxins element and proper transducer depends on the properties of the sample of interest and the physical magnitude to be measured. The recognition element, that is, the biocomponent, determines the degree of selectivity or specificity of the final biosensor. In this chapter, a brief commentary on some aspects of biosensor construction is reported. Current situation, recent development, and applications of biosensors for food technology in Brazil and Italy are reviewed.

11 Recent Biosensors for Food Analysis in Brazil and Italy 167 11.2 Biosensor for Small Molecule Determination in Food Analysis Among proposed biosensors, electrochemical transduction systems were the most used. Among them, amperometric and potentiometric transduction have found the widest applications, even if other transducers are available. The combination of oxidoreductases, as recognition bioelements, and amperometric electrodes, as trasducers, gave good results for food analysis, mainly because enzymatic activity, depending on substrate concentration in food samples, can be easily measured with reasonable sensitivity. This combination constitutes one of the most successful classes of biosensors. Recently, Ferreira et al. (2004) immobilized two enzymes, namely, β-galactosidase and glucose oxidase, in order to determine lactose in cheese whey. The biosensor was based on the determination of oxygen consumption, which occurs during the enzymatic reaction. Authors studied the influence of temperature on the biosensor signal, observing a nonlinear relationship between the biosensor electric response and lactose concentrations a function of temperature and analyte concentration. This was due to differences in temperature dependencies of enzyme activities. Nonlinear correlations were proposed to automatically com- pensate the effects of temperature. Mello et al. (2003) developed a biosensor based on horseradish peroxidase (HRP) and immobilized DNA onto silica–titania and applied the novel system to the measurement of polyphenol compounds in vegetables samples. Various analytical parameters influencing the biosensor performances were studied as a function of chlorogenic acid, as reference polyphe- nol compound. The effect of working potential, type and concentration of the buffer, pH, response time, and interferences was investigated. The biosensor showed a linear response in the range from 1 to 50 μM chlorogenic acid, applying a potential of À50 mV versus Ag/AgCl, with a sensitivity of 181 nA/μM/cm2 and a detection limit of 0.7 μM. The biosensor was tested for polyphenol determination in vegetable extract and the results were compared with the Folin–Ciocalteau tradi- tional method. The biosensor showed suitability to the quantification of the total polyphenol in the tested samples. Other authors described a highly selective and stable electrochemical biosensor for the determination of glucose in soluble coffee (de Mattos and Areias 2005) The biosensor electrode consisted of a thin film of ferric hexacyanoferrate, electrodeposited on the glassy carbon electrode and glucose oxidase immobilized on top of the electrode surface. Stability of the thin film was evaluated by injecting standard solution of H2O2 and glucose during 4 h in a flow-injection system, with the electrode polarized at À50 mV versus Ag/AgCl. The system was able to handle about 60 samples per hour, with high stability, and was proposed for industrial process control. A linear calibration in the range of 0.15 and 2.50 mM glucose and a detection limit of 0.03 mM were obtained. Another biosensor for glucose determi- nation in food samples was developed by one of the authors (Baratella et al. 2013). The experimental work demonstrated the peculiar electro-catalytic behavior of a new generation of iron oxide nanoparticles (surface-active magnetite nanoparticles,

168 V.A. Pedrosa et al. SAMNs), which were used for the development of a cheap carbon paste electrode aimed at hydrogen peroxide detection, containing an ionic liquid, namely, 1-butyl- 3-methylimidazolium hexafluorophosphate (BMIM-PF6), and characterized by a sensitivity of 206.51 μA/mM/cm2, a detection limit of 0.8 μM H2O2, and a noise of 1.01 nA. Furthermore, these metal oxide nanoparticles were used to form stable conjugates with rhodamine isothiocyanate, acting as a bridge, permitting the cova- lent binding of glucose oxidase. The resulting bio-conjugate was used to develop a nanocomposite, carbon paste-BMIM-PF6-based biosensor, characterized by a sen- sitivity of 48 μA/mM/cm2, in the 0–1.5 mM glucose concentration range, and a detection limit of 0.9 μM glucose. The system was tested on fruit juices as real samples, without any sample preparation procedure and results suggest that BMIM- PF6–(SAMN@RITC–GOx)/CPE biosensor could be a promising, low-cost option for the development of GOx-based biosensors for glucose determination. Amati et al. (2008) reported on a biosensor for the determination of the total antioxidant capacity and the total polyphenol concentration in extra virgin oil (EVO), as well as the main kinetic parameters of the process during the thermal oxidation of EVO. They also evaluated the increase of radical concentration during the thermal oxidation process, using a superoxide dismutase biosensor. The inves- tigation concerning this important food product was of high interest, as it referred to the state of alteration of the EVO, used for cooking or frying, as a function of temperature. Currently, the food industry is very receptive to biosensor technology, while a new market will probably be developed in the long run. A method for the rapid detection of common compounds will probably offer the best opportunity for biosensors in this industrial field, but several key issues also need to be resolved before biosensors find widespread applications. Newly, a new biosensor for the direct determination of lactic acid and malic acid in wines was developed (Mazzei et al. 2007). This multi-enzymatic biosensor was realized for the selective determi- nation of three analytes: D(À) and L(+)-lactic acid and L(À)-malic acid. The measurement was based on a multi-enzymatic biosensor employing the catalytic activities of L(+)-lactate oxidase, D(À)-lactate dehydrogenase, and horseradish peroxidase for the determination of total D(À)- and L(+)-lactic acid and a bienzymatic electrode for L(À)-malic acid determination, realized by coupling the L(À)-malic dehydrogenase and horseradish peroxidase. For both electrodes, enzymes were immobilized on an oxygen-selective Clark electrode and the simul- taneous determination of the two organic acids was accomplished either in batch or in a flow injection analysis apparatus, using the same biosensors as detectors. The analytical performances were tested in standard aqueous solutions and on real wine samples, showing high repeatability, short response times, and reduced cost of analysis, suggesting that the experimental approach here described could be conve- nient to monitor the progress of malo-lactic fermentation in wines. A paper by Centonze and coworkers described a biosensor for simultaneous monitoring of glucose and ethanol content in drinks and alcoholic fermented media (Mentana et. al 2013). The methodology was based on the immobilization of glucose oxidase and alcohol oxidase by co-cross-linking with bovine serum

11 Recent Biosensors for Food Analysis in Brazil and Italy 169 albumin and glutaraldehyde onto a dual gold electrode, modified with a perm selective over oxidized polypyrrole film. The biosensor was integrated in a flow injection system, coupled with an online microdialysis fiber as sampling tool. Flow rates inside and outside the fiber were optimized in terms of linear responses (0.01–1 and 0.01–1.5 M) and sensitivities (27.6 Æ 0.4 and 31.0 Æ 0.6 μA/mM/ cm2) for glucose and ethanol, respectively. Excellent anti-interference characteristics, with total absence of “cross talk,” and good response stability, under the operational conditions, allowed the application of the dual biosensor to the accurate real-time monitoring of alcoholic drinks and white grape musts. Arecchi et al. (2010) described an amperometric biosensor for the detection of phenolic compounds in food, based on tyrosinase as bioelement. The enzyme was immobilized by drop-coating on a glassy carbon electrode, covered by a polyamidic nanofibrous membrane, prepared by electrospinning. With respect to others examples in the literature, the selectivity of the tyrosinase biosensor resulted in modification by the presence of the nanostructured coating, which seemed to affect the permeability of polyphenols as a function of solution pH, depending on poly- phenol dissociation constants. The biosensor exhibited a response time of 16 s, a detection limit of 0.05 μM, and a linearity up to 100 mM. This biosensor was successfully used for real-time monitoring of the release kinetics of phenols, encapsulated in polymeric microcapsules. Moreover, innovative detection methods for toxic compound detection in foods were proposed by different groups in Brazil and Italy. An example dealt with the development of a biosensor as an analytical device for the detection of beta-lactam residues in milk (Ferrini et al. 2008). This indirect method was based on the measurements of carbon dioxide (CO2), produced by the microbial growth of a reference microorganism, namely, Bacillus stearothermophilus var. calidolactis. The addition of milk samples to the cultivation medium led to microbial growth inhibition, if beta-lactams are present, and, consequently, a decrease of CO2 production rate. The analysis was based on the differences of CO2 production between a milk sample, spiked with beta-lactams, and a twin milk sample, containing beta-lactams plus a broad spectrum beta-lactamases, using an electro- chemical device. Moreover, the ability to sense all of the beta-lactams speeds the total time of analysis, when chemical identification and quantification are not required. The analytical method was adequate for milk control for qualitative screening purposes, complying with the requirements stated in Europe by the Decision 2002/657/EC. Campanella et al. (2009) described a new biosensor for rapid determination of nonsteroidal anti-inflammatory drugs (NSAIDs), based on the inhibition of cyclooxygenase by NSAIDs in fresh milk. The results showed the full validity of the method, which was optimized by comparing the inhibition of two enzyme isoforms, COX-1 and COX-2, in the presence of different tested pharma- ceutical drugs (diclofenac, naproxen, ibuprofen, tolmetin). Recovery trials were performed in adulterated milk and fresh cheese samples with known concentrations of NSAIDs, always obtaining recovery values >88 %. To date control strategies in detecting anabolic agents for promoting growth of food producing animals are mainly related to screening techniques based on immunochemical and physiochemical methods, whose major limit is represented

170 V.A. Pedrosa et al. by relative low analytical sensitivity. As a consequence, consumers are currently exposed to molecules with potential carcinogenic effects, such as 17β-estradiol, the most powerful substance with estrogenic effect. Therefore, high analytical sensitiv- ity screening and confirmatory methods are required, coupling easiness of use and efficiency. Ricciardi et al. (2013a, b) reported on the immunodetection of 17- β-estradiol in serum by antibody-immobilized microcantilever resonators, an inno- vative biosensing platform able to quantify an adsorbed target mass (such as cells, nucleic acids, biomolecules, etc.) thanks to a shift in resonance frequency. The analytical tool showed to be capable of discriminating treated and untreated animals, showing the ability of detecting traces of 17β-estradiol in serum at concentrations lower than the present accepted physiological serum concentration threshold value (40 ng/kg) and commercial ELISA tests (25 ng/kg). The method exhibits a limit of detection of 20 ng/kg and a limited cross-reactivity with high concentrations (10 μg/kg) of similar molecules (testosterone). 11.3 Biosensors for Bacteria and Bacteria Toxins Detection The presence and diffusion of pathogenic bacteria in foodstuff represent the main concern for food safety, and innovative determination methods for microorganism and biological toxin detection, based on biosensor technology, were proposed. Recently, Rejeb et al. (2009) reported on a biosensor based on acetylcholinesterase (AChE) inhibition by mycotoxins, namely, aflatoxin B-1 (AFB-1). AChE was present in solution and an amperometric choline oxidase biosensor was used for monitoring AChE residual activity by determining choline produced from acetyl- choline hydrolysis. To create the biosensor, choline oxidase was immobilized by cross-linking onto a screen-printed electrode modified with Prussian Blue and this was used to detect the H2O2 produced by choline oxidation at low applied potential (À0.05 V versus a screen-printed internal silver pseudo-reference electrode). For the development of the AFB-1 assay, several parameters, such as AChE and substrate concentrations, the effect of methanol, and pH, were evaluated and optimized. Authors found a linear working range of 10–60 ppb for AFB-1, and concentrations as low as 2 ppb, corresponding to the legal limit of AFB-1 in food for humans, were detected, after a pre-concentration step. The suitability of the method was evaluated using commercial olive oil samples. Reis group’s reported a new methodology based on the colorimetric response induced by pathogenic bacteria (Staphylococcus aureus and Escherichia coli) (Pires et al. 2011). The addition of bacterial supernatants caused a colorimetric modification in the presence of 10,12-pentacosadyinoic acid (PCDA) and N- [(2-tetradecanamide)-ethyl]-ribonamide (TDER) vesicles, even in diluted concentrations, indicating that chemical interactions occur between the vesicles and bacteria. It was observed that bacterial substrates released from S. aureus induced a more intense color change, compared to the optical response induced by E. coli. S. aureus metabolites also induced a more pronounced color change when TDER/PCDA vesicles were incorporated into cellulose strips. Authors analyzed the colorimetric response in the presence of interferent molecules, using

11 Recent Biosensors for Food Analysis in Brazil and Italy 171 apple juice as food matrix. Both apple juice samples, sterile and inoculated with bacteria, induced a TDER/PCDA color change; however, the S. aureus supernatants induced a slightly greater color response both in the suspensions and in the cellulose strips. TDER/PCDA vesicles showed a great potential for the development of biosensors to detect food pathogens in intelligent food packaging. A simple and fast response biosensor for screening nisin, directly identifying nisinogenic bacteria, by bioluminescence detection of Lactococcus lactis was proposed (Virolainen et al. 2012). The method is based on the nisin-controlled gene expression system which facilitates efficient overexpression of heterologous genes. The overlay of putative nisinogenic colonies with the biosensor strain gives identification results within 1 h. Functionality and specificity of the method were verified by screening for the presence of nisin producing bacteria among 144 raw milk colonies and a panel of 91 lactococcal strains. Studies performed on strains and colonies, which did not induce bioluminescence but inhibited Lactococcus lactis NZ9800lux growth, demonstrated that only nisinogenic bacteria can cause bioluminescence induction. Bacteria known to produce bacteriocins, other than nisin, failed to induce bioluminescence, further confirming the specificity of the assay. Moreover, authors discovered a new non-inducing, but inhibitory, lactococcal strain harboring a modified nisin Z gene and demonstrated that the source of the inhibitory action is not a non-inducing variant of nisin, but a bacterio- cin of lower molecular weight. The concentration of nisin producing bacteria in a raw milk sample was 1.3 Â 102 CFU/mL. A total of seven nisin Z producing colonies of L lactis subsp. lactis, which were shown to belong to three different groups by genetic fingerprinting, were also identified in raw milk samples. The presented biosensor is robust, cost-effective, and simple to use, avoiding the pitfalls of traditional screening methods by directly specifying the identity of the toxic substance. Zamolo et al. (2012) developed an ultrasensitive electrochemiluminescence- based sensor for the detection of palitoxin (PlTX), one of the most potent marine toxins, frequently detected in seafood, taking advantage of the specificity provided by anti-PlTX antibodies, the good conductive properties of carbon nanotubes, and the excellent sensitivity achieved by a luminescence-based transducer. The sensor was able to produce a concentration-dependent light signal, allowing PlTX quanti- fication in mussels, with a limit of detection of 2.2 μg/kg of mussel meat), more than two orders of magnitude more sensitive than that of the commonly used detection techniques, such as LC-MS/MS. An antibody-immobilized microcantilever resonator system was proposed for the detection of mycotoxins, such as aflatoxins and ochratoxin A, which are considered as the most important chronic dietary risk factor, more than food additives or pesticide residues (Ricciardi et al. 2013a, b). The feasibility of using microcantilever resonator arrays to effectively identify total aflatoxins and ochratoxin A, at low concentrations (3 ng/mL and less than 6 ng/mL, respectively), with relatively low uncertainty (about 10 %) and good reproducibility for the same target concentration, was shown. Furthermore, the developed immunosensing method shows a limited cross-reactivity to different mycotoxins, paving the way to a highly specific technique, able to identify different mycotoxins in the sample.

172 V.A. Pedrosa et al. Surface plasmon resonance (SPR)-based DNA biosensors were shown to be rapid, label-free, and selective tools for the detection of PCR products (Pascale et al. 2013). An SPR sensor based on DNA hybridization for the detection of Fusarium culmorum, a fungal pathogen of wheat, was described. A 0.57 kb DNA fragment of F. culmorum was amplified by specific primers, and a 25-mer oligonu- cleotide probe was selected within the sequence of the PCR amplified. The biotin- labeled probe was immobilized on a streptavidin sensor chip and tested for biospecific interaction with PCR products of F. culmorum. The SPR biosensor was applied to the detection of F. culmorum in fungal cultures and in naturally infected wheat samples. Another example from Italy dealt with an electrochemical immunoassay, devel- oped using magnetic beads as solid phase and carbon screen-printed arrays as transducers, was developed for the detection of sulfonamides in food matrices (Centi et al. 2010). Magnetic beads, coated with protein A, were modified by immobilization of specific antibodies and a competition between the target analyte and the corresponding labeled analyte was carried out. Analyte labeling was performed with alkaline phosphatase. After the immunosensing step, beads were captured by a magnet onto the working electrode surface of a screen-printed eight- electrode array for a multiple electrochemical detection. Screen-printed eight- electrode arrays were chosen as transducers due to the possibility to repeat multiple analysis and to test simultaneously different samples. The determination was performed by differential pulse voltammetry, as fast electrochemical technique. Calibration curves demonstrated that the developed electrochemical immunoassay was able to detect concentrations as low as ng/mL. The short incubation times and the fast electrochemical measurement make this system a possible alternative to classic ELISA tests. Nucleic acid aptamers have been presented as a new way to detect pathogenic compound. These macromolecules have attracted intense interest and found wide applications in a range of areas (Palchetti and Mascini 2012; Tombelli and Mascini 2009). Aptamers exhibit many advantages as recognition elements in biosensing when compared to traditional antibodies. They are small in size, chemically stable, and cost effective. More importantly, aptamers offer remarkable flexibility and convenience in the design of their structures, which has led to novel biosensors that have exhibited high sensitivity and selectivity. Recently, Castillo et al. (2012) development a biosensor based on DNA aptamers for detection of ochratoxin A (OTA). The thiolated DNA aptamers specific to OTA of different configurations have been immobilized by chemisorption to the surface of a gold electrode. Electrochemical impedance spectroscopy in the presence of a redox probe, such as [Fe(CN)6]À3/À4, has been used for the determination of the charge transfer resistance, following the addition of OTA containing samples. Charge transfer resistance increased with increasing OTA concentration in the range 100–0.1 nM. The limit of detection (0.12 nM) depended on aptamer configuration. The sensor was renewable and validated on food samples with satisfactory recovery. Silva et al. (2012) described a new DNA biosensor for the detection of toxigenic Penicillium sclerotigenum in pure culture or infected yams. The P. sclerotigenum detection takes place on a self-assembled monolayer of a (magnetite)/(poly

11 Recent Biosensors for Food Analysis in Brazil and Italy 173 (allylamine hydrochloride) (Fe3O4–PAH) composite that serves as an anchoring layer for the DNA hybridization interaction. Electrical impedance spectroscopy (EIS) was used to evaluate and quantify the hybridization degree. The Fe3O4–PAH composite represents a good platform for the immobilization of biomolecules, due to the presence of many possible binding sites for nucleotides and to its large surface-to-volume ratio, and good biocompatibility. The biosensor was capable of not only qualitatively detecting the presence of the fungus genome at low concentrations, but also showed a good quantitative impedimetric response. A Fe3O4–PAH-probe biosensor would require only small volumes and low concentrations of the analyte when used, for instance, in detecting P. sclerotigenum contamination of food, besides presenting many competitive advantages, such as selectivity, specificity, and reproducibility, relative to alterna- tive techniques. The development of a novel electrochemical immunosensor for the sensitive detection of staphylococcal enterotoxin A (SEA) based on self-assembly monolayer (SAM) and protein A immobilization on gold electrode was reported (Pimenta- Martins et al. 2012). Three different methods of protein A immobilization were tested: physical adsorption, cross-linking using glutaraldehyde, and covalent binding after activation with N-hydroxysuccinimide/N-ethyl-N0-(3-dimethylaminopropyl) carbodiimide hydrochloride on cysteamine-modified gold electrode. The EDC/NHS method for protein A immobilization was selected to lead development of the biosensor. The coating steps of the surface modification were characterized by cyclic voltammetry and the biosensor response by chronoamperometry. The advantages of the immunosensor were exposed in its high sensitivity and specificity. The proposed amperometric immunosensor was successfully used for determination of SEA in contaminated and non-contaminated cheese samples. 11.4 Pesticide Biosensors Pesticides represent another class of compounds that have been studied in food analysis by different research groups in Brazil and Italy. Pesticides have been associated with many health hazards and rapid and reliable monitoring of these contaminants is mandatory. Traditional chromatographic methods, such as high-performance liquid chroma- tography and capillary electrophoresis, equipped with mass spectrometry detectors, are effective for the analysis of pesticides in the environment, but present previ- ously cited limitations. Thus, the use of biosensors to replace classical analytical methods by simplifying or eliminating sample preparation and making field-testing easier and faster with significant decrease in cost per analysis is attractive (Kantiani et al. 2010; McGrath et al. 2012). A sensitive electrochemical acetylcholinesterase (AChE) biosensor was success- fully developed with polyaniline (PANI) and multi-walled carbon nanotubes (MWCNTs) core–shell modified glassy carbon electrode (GC) and used to detect carbamate pesticides in fruit and vegetables (apple, broccoli, and cabbage).

174 V.A. Pedrosa et al. The pesticide biosensors were applied in the detection of carbaryl and methomyl pesticides in food samples using chronoamperometry. The GC/MWCNT/PANI/ AChE biosensor exhibited detection limits of 1.4 and 0.95 μM, respectively, for carbaryl and methomyl. These detection limits were below the allowable concentrations set by Brazilian regulation standards for the samples in which these pesticides were analyzed. Reproducibility and repeatability values of 2.6 % and 3.2 %, respectively, were obtained with the procedure. The proposed biosensor was successfully applied for the determination of carbamate pesticides in cabbage, broccoli, and apple samples, without any spiking procedure. The obtained results were in full agreement with those from HPLC analysis (Cesarino et al. 2012). Pedrosa et al. (2007) reported on an acetylcholinesterase (AchE)-based ampero- metric biosensor developed by immobilization onto a self-assembled modified (SAM) gold electrode. Cyclic voltammetric experiments performed with the SAM-AchE biosensor in phosphate buffer solutions, containing acetylthiocholine, confirmed the formation of thiocholine and its electrochemical oxidation at +0.28 V vs Ag/AgCl. An indirect methodology involving the inhibition effect of parathion and carbaryl on the enzymatic reaction was developed and employed to measure pesticides in food samples without pretreatment or pre-concentration steps. Values higher than 91–98.0 % recovery indicated the feasibility of the proposed electroan- alytical methodology to determine pesticides in food samples. The results obtained by the biosensor were compared with HPLC measurements, confirming the amperometrically measured values. The same research group reported on the detection of carbamates (a common class of pesticides in Brazil) in different vegetables (Cesarino et al. 2012). An electrochemical acetylcholinesterase biosen- sor was successfully developed on polyaniline and multi-walled carbon nanotubes core–shell modified glassy carbon electrode. The pesticide biosensor was applied to the detection of carbaryl and methomyl pesticides in food samples, by chronoam- perometry. The biosensor exhibited detection limits of 1.4 and 0.95 μM for carbaryl and methomyl, respectively, which were below the allowable concentrations set by Brazilian regulation standards. Reproducibility and repeatability values of 2.6 % and 3.2 %, respectively, were obtained, and the proposed biosensor was success- fully applied on cabbage, broccoli, and apple samples, without any spiking proce- dure. The obtained results were in full agreement with those obtained by HPLC. Other paper describes the development of methodology for carbaryl determina- tion in tomatoes (Caetano and Machado 2008). The measurements were carried out using an amperometric biosensor based on the inhibition of acetylcholinesterase activity due to carbaryl adsorption. The analytical curve obtained in buffered solutions showed excellent linearity in the 5.0 Â 10À5 to 75 Â 10À5 M range, with a limit of detection of 0.4 Â 10À3 g/L. The application of the developed methodology on tomato samples involved a simple sample solubilization, followed by carbaryl spiking at different concentrations. Recovery values were in the 92.4–99.0 % range. For comparison, HPLC experiments were also carried out under similar conditions. However, with this analytical procedure, tomato samples have to be manipulated by an extraction procedure, which yielded much lower recovery values (78.3–84.8 %). Finally, the biosensor was employed to analyze

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Chapter 12 Natural Ingredients as Additive for Active Antioxidant Food Packaging Carolina Oliveira de Souza, Pricila Veiga-Santos, and Janice Izabel Druzian Abstract Active packaging materials have been developed to interact with the packed products and to extend their shelf life. When regarding antioxidant active packaging, it also has the advantage of allowing lower antioxidant addition to the food product. Although many synthetic antioxidant have already been used as antioxidant additives for polymers, recent studies have demonstrated that natural antioxidants are promising sources of additives. In this chapter, some aspects related to natural antioxidants have been reviewed and a few natural antioxidant sources, used as additive for active antioxidant food packaging materials, have been discussed. Keywords Antioxidant • Active packaging • Natural additives 12.1 Introduction Packaging materials are traditionally used to hold, protect, and sell food products. The protecting layer has the aim of preserving quality in order to minimize physical, chemical, and biochemical alterations that might contribute to the product degradation. The food industry, seeking for competitive advantages, search for safe and high-quality products. In this matter, packaging systems have been studied with the objective to interact with the packaged food, helping to extend shelf life (Azeredo et al. 2000). Such packaging materials are called “active packagings.” The term “active” has been applied for the first time to food by Ted Labuza, in 1987, in a Scottish Conference about the nutritional impact of processed foods C.O. de Souza • J.I. Druzian 179 Faculty of Pharmacy, UFBA, 40170.970 Salvador, BA, Brazil P. Veiga-Santos (*) UNESP – Universidade Estadual Paulista, 18610.307 Botucatu, SP, Brazil e-mail: [email protected] G.P.P. Lima and F. Vianello (eds.), Food Quality, Safety and Technology, DOI 10.1007/978-3-7091-1640-1_12, © Springer-Verlag Wien 2013

180 C.O. de Souza et al. (Rooney 2005) and the article that literally applied the term “active packaging” was first published in December of 1986, entitled “Alcan Micro Match: an active packaging system” by Smith, J.D. (Mendes 2010). Among the most important active packaging materials is the antioxidant pack- aging, which has a protective effect against the oxidation of the packed product (Vermeiren et al. 1999; Lo´pez-de-Dicastillo et al. 2012). Its utilization in food can also allow the production of food with lower antioxidant addition (Rooney 1995; Soares and Hotchkiss 1998; Hayashi et al. 2006; Souza et al. 2011), helping to avoid allergies related to food preservative ingestion (Ahvenainen 2003). Among the many existing antioxidant additives, those from natural sources have been cited as promising substitutes for synthetic additives in packaging materials (Hayashi et al. 2006; Grisi et al. 2008; Souza et al. 2011). In this work, some aspects related to natural antioxidants have been considered and a few natural antioxidant sources, used as additive for active antioxidant packaging of food materials, have been discussed. 12.2 Natural Antioxidants Halliwell and Gutteridge (1995) defined antioxidants as “any substance that, when present at low concentrations compared with that of a substrate that can be oxidized, significantly delays or inhibits oxidation of that substrate,” but later defined them as “any substance that delays, prevents or removes oxidative damage to a target molecule” (Halliwell 2007). The antioxidant activity can be effective through various ways: as inhibitors of free radical oxidation reactions (preventive oxidants), by inhibiting formation of free lipid radicals; by interrupting the propagation of the autoxidation chain reac- tion (chain breaking antioxidants); as singlet oxygen quenchers; by synergism with other antioxidants; as reducing agents, converting hydroperoxides into stable compounds; as metal chelators, transforming metal pro-oxidants (iron and copper derivatives) into stable products; and finally as inhibitors of pro-oxidative enzymes (lipo-oxygenases) (Kancheva 2009; Carocho and Ferreira 2012). The oxidative deterioration of fats and oils in foods is responsible for rancid odors and flavors, with a consequent decrease in nutritional quality and safety, caused by the formation of secondary, potentially toxic compounds. The addition of antioxidants is required to preserve food flavor and color, and to avoid vitamin destruction. Among synthetic antioxidants, the most frequently used to preserve food are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), and tert-butyl hydroquinone (TBHQ). Reports reveal that BHA and BHT could be toxic, and their high manufacturing costs, along with the increasing consciousness of consumers with regard to food additive safety, created a need for identifying a more natural, and probably safer, antioxidant alternative (Moure et al. 2001; Bonilla et al. 2012). The replacement of synthetic antioxidants by natural ones may have benefits due to health implications and functionality of food systems, such as solubility in both

12 Natural Ingredients as Additive for Active Antioxidant Food Packaging 181 oil and water, which is of interest for application in emulsions (Moure et al. 2001). Vegetable materials contain many compounds with antioxidant activity. Several plants (seeds, fruits, leaves, and roots) and derivatives have been studied as sources of potentially safe natural antioxidants for the food industry. Among the antioxidant components most thoroughly investigated from vegetables sources, polyphenols, flavonoids, carotenoids, vitamins, organic acids, and tocopherols are the most studied (Kaur and Harish 2001; Oliveira et al. 2011). Polyphenols are secondary plant metabolites and confer both desirable and undesirable food qualities to fruits and vegetables. They are ubiquitous in plant material and sometimes present as esters and glycosides, possessing antioxidant activity as chelators and free radical scavengers, with special impact over hydroxyl and peroxyl radicals, superoxide anions, and peroxynitrites. One of the most studied and promising compounds, belonging to the hydroxybenzoic group, is gallic acid, which is also the precursor of many other tannins, while cinnamic acid is the precursor of all the hydroxycinnamic acids (Krimmel et al. 2010). Flavonoids and related compounds occur in many plant and fruits. They belong to an antioxidant group of compounds, composed of flavonols, anthocyanins, isoflavonoids, flavanones, and flavones. Their antioxidant properties are conferred on the phenolic hydroxyl groups, attached to aromatic ring structures, and they can act as reducing agents, hydrogen donators, singlet oxygen quenchers, superoxide radical scavengers, and even as metal chelators. They are also able to activate antioxidant enzymes, reduce α-tocopherol radicals (tocopheroxyls), inhibit oxidases, mitigate nitrosative stress, and increase levels of uric acid and other low molecular weight molecules. Some of the most important flavonoids are catechin, catechin-gallate, quercetin, and kaempferol (Prochazkova et al. 2011). Carotenoids are a group of natural pigments that are synthesized by plants and microorganisms, but not by animals. They are frequently used as natural coloring materials, but they also possess antioxidant activity, especially in the presence of light. The main antioxidant property of carotenoids is against singlet oxygen. They can be separated into two vast groups: the carotenoid hydrocarbons, known as the carotenes, which contain specific end groups, such as lycopene and β-carotene, and oxygenated carotenoids, known as xanthophylls, such as zeaxanthin and lutein (Carocho and Ferreira 2013). Many studies have evidenced the antioxidant potential of natural plants, vegetables, oils, fruits, teas, etc., and in Table 12.1 a list of some natural compounds with antioxidant activity is presented. Studies about the use of these natural antioxidants, as food additives, have increased in last years, and results are encouraging.

182 C.O. de Souza et al. Table 12.1 Antioxidants from natural sources Sources of active References compounds Mango/acerola Investigation on the antioxidant activity of leaves, peels, stems bark, and kernel of mango (Mangifera indica L.) (Sultana et al. 2012) Palm oil/ac¸a´ı Agronomic characterization and antioxidant potential of fruit from clones Oregano oil/yerba of the acerola plant (Cunha et al. 2012) mate tea Phenolic acid analysis and antioxidant activity assessment of oil palm (E. Wine/jabuticaba guineensis) fruit extracts (Neo et al. 2010) Cocoa/coffee Ac¸a´ı (Euterpe oleraceae) “BRS Para´”: A tropical fruit source of antioxi- dant dietary fiber and high antioxidant capacity oil (Rufino et al. 2011) Sensory attribute preservation in extra virgin olive oil with addition of oregano essential oil as natural antioxidant (Asensio et al. 2012) Antioxidant activity of yerba mate extracts: Interactions between the individual polyphenols (Valerga et al. 2013) Prediction of total antioxidant capacity of red wine by Fourier transform infrared spectroscopy (Versari et al. 2010) Jaboticaba peel: Antioxidant compounds, antiproliferative and antimutagenic activities (Leite-Legatti et al. 2012) Comparison of the antioxidant activity of commonly consumed polyphe- nolic beverages (coffee, cocoa, and tea) prepared per cup serving (Richelle et al. 2001) 12.3 Antioxidant Food Packaging Incorporation of antioxidants into packaging films has become very popular, since oxidation was recognized as one of the main causes of food spoilage. Oxidation alters the taste (rancidity) and nutritional quality (loss of vitamins and essential fatty acids) of foods, and generates reactive and toxic compounds, which may represent a danger to consumers (Laguerre et al. 2007). Synthetic antioxidants are the most used antioxidant additives to prevent/retard the oxidation process. Such additives recently received a great deal of interest due to toxicological concerns, prompting an increased interest in natural antioxidants, such as those derived from fruits, vegetables, plants, and others (Bonilla et al. 2012). Recently, researches about the applications of natural antioxidants in active packaging have being cited in the literature, as reported in Table 12.2. Antioxidants properties of protein-based films from fish skin gelatin, incorporated with different citrus essential oils, including bergamot, kaffir lime, lemon, and lime, were investigated. Films incorporated with lemon essential oil showed the highest ABTS radical scavenging activity and ferric reducing antioxi- dant power, among other modified films (Tongnuanchan et al. 2012). Souza et al. 2011 developed several active films from starch cassava, containing mango and acerola pulps as antioxidant additives, using a response surface meth- odology for film characterization. The films were used to pack palm oil (maintained for 45 days of storage) under accelerated oxidation conditions (63 % relative

12 Natural Ingredients as Additive for Active Antioxidant Food Packaging 183 Table 12.2 Natural antioxidant additives for active packaging materials Antioxidante Based films Packaged Results References natural product Green tea extract Chitosan The incorporation of aque- Siripatrawan Gelatin ous green tea extract and Harte into chitosan films (2010) improved mechanical Wu and water vapor barrier et al. (2013) properties and enhanced polyphenolic content and antioxidant activity of the films The incorporation of GTE into gelatin films enhanced the total phe- nolic content, DPPH radical scavenging activity, and reducing power. However, DPPH radical scaveng- ing activity and reduc- ing power decreased during storage Essential oils of Milk protein Beef muscle Oregano-based films Oussalah pimento and oregano stabilized lipid oxida- et al. (2004) tion in beef muscle samples, whereas pimento-based films presented the highest antioxidant activity Marigold extract Low-density Soybean oil Spectroscopic, optical, and Col´ın‐Cha´vez polyethylene mechanical properties et al. (2012) of the films were affected by the addition of the marigold extract. However, bags made of the films showed a positive effect on soy- bean oil stability when used as packaging Barley husks Low-density Blue shark The results confirm the Pereira de polyethylene muscle efficacy of active pack- Abreu aging with a natural et al. (2011) antioxidant derived from barley husks to slow the progress of lipid hydrolysis and increase oxidative sta- bility in blue shark muscle Mango and acer- Starch Palm oil Although the film-forming Souza ola pulps procedure affected the et al. (2011) (continued)

184 C.O. de Souza et al. Table 12.2 (continued) Antioxidante Based films Packaged Results References natural product antioxidant compounds, the results indicated that antioxidants were effective additives for protecting the packaged product Palm fruit Cassava starch Soybean oil Results have indicated that Grisi the palm antioxidant et al. (2008) compounds in the packaging material were preferentially oxidized, lowering the oxidation of the packed product Cocoa/coffee Cassava starch Palm oil Results have indicated Silva (2009) efficacy when protecting the product in accelerated storage conditions. Besides the antioxidant effect, the resulting films also were colored by the cocoa and coffee pigments and flavored by their pleasant natu- ral flavors Barley husks Low-density Blue shark The results confirm the Pereira de polyethylene muscle efficacy of active pack- Abreu aging with a natural et al. (2011) antioxidant derived from barley husks to slow the progress of lipid hydrolysis and increase oxidative sta- bility in blue shark muscle Mustard meal Xanthan gum Smoked The composite coating Kim salmon improved the stability et al. (2012) of smoked salmon against lipid oxidation without imparting a negative sensory qual- ity to the salmon humidity and 30 C) to simulate a storage experiment. Bio-based films were prepared (casting) by dispersing cassava starch (4 %), sucrose (1.4 %), inverted sugar (0.7 %), and mango and/or acerola fruit pulp (0–20 %) in distilled water,

12 Natural Ingredients as Additive for Active Antioxidant Food Packaging 185 according to a (22) second-order experiment design (for a total of 11 experiments). It was noted that palm oil packed in bio-based films containing mango and acerola pulps showed a low peroxide index, when compared to the product packed in control films. The results indicated the efficacy of fruit pulps as antioxidant additives, acting to protect the packaged product. This effect can be considered concentration-dependent; palm oil packed in films with low pulp concentrations presented a higher oxidation value (IP ¼ 64.27 %), when compared to packed in films with higher pulp concentrations, showing a lower PI value (IP ¼ 31.62 %), during the same storage period. The increase of carotenoid content in the film showed a higher correlation with peroxide index (98.39 %) than polyphenols (56.99 %), confirming the efficacy of carotenoids incorporated into films in com- parison to polyphenols. As mango pulp possesses a higher amount of carotenoids than acerola pulp, the product packed in films containing mango pulp showed less oxidation. It was observed by authors that the packaging material, rather than the packaged product, was oxidized, due to the active compound loss during storage. Figure 12.1 shows the mango and acerola antioxidant film and the surface response of the increase in the peroxide value of the packed oil after 45 days storage (63 % relative humidity and 30 C), influenced by the mango and acerola pulp concentra- tion as additive. A similar behavior was observed in films containing palm fruit pulp and its oil, as added antioxidants, which were used to pack soybean oil. In this case, a decrease in TC ranging from 79.90 to 99.60 % was observed during 90 days of storage (Grisi et al. 2008). Active polymers added to palm oil had presented the best antioxidant effect in packed oil (64 % RH, 30 C). Authors noticed that increasing the palm fruit derivates’ (extract and oil) addition decreased the final film total carotenoid content. However, when observing the total carotenoid content in the packed product, the higher was the antioxidant addition to the film, especially palm oil, the higher was the total carotenoid content. Results have indicated that, also in this study, the packaging material was oxidized, rather than the packaged product. Besides the presence of fibers, resulting from palm fruit addition, and oil incorporation, the polymer appeared with good transparency. Figure 12.2 shows the film appear- ance and transparency after palm fruit addition, and the surface response surface of the palm fruit extract vs. palm oil effect on the total carotenoid content (TC) of the packed product (soy bean oil) after 90 days of storage (63 % relative humidity and 30 C). 12.4 Conclusions Literature results have demonstrated that natural antioxidants can be incorporated, as additives, in conventional or biodegradable active packaging materials. Unfor- tunately, food and pharmaceutical industries have taken little advantage of such an innovation opportunity. A possible reason can be the lack of studies about large- scale production of such antioxidant materials.

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Index A effect of GPs, 113–115 Ac¸a´ı fruits. See Euterpe oleracea quantitative real-time RT-PCR, Acidified sodium chlorite (NaClO2), 47 Actinobacteria, 148–149 112–120 Actinomyces RNA-Seq, 122 Anacardium occidentale, 6, 11 cellulase, 153 Aniba parviflora, 11 extracellular peptidases, 154 Antibody-immobilized microcantilever lipases, 156 taxonomy, 148–149 resonator system, 171 Actinomycetales, 149 Anti-inflammatory effects Active food packaging advantages, 180 Baccharis trimera, 11 definition, 179–180 lycopene, 72 natural antioxidants Saratudo, 10 Streptomyces, 149–150 additives, 182–185 Antimicrobial solutions, 46–47 antioxidant activity, 180 Antioxidants carotenoids, 181 activity, measurement of, 5 flavonoids, 181 additives, natural, 183–184 polyphenols, 181 carotenoids, 5 sources, 182 definition, 4 vs. synthetic antioxidants, 180–181 in fruits palm fruit film appearance, 185, 186 synthetic antioxidants, 180–182 Annona crassifolia, 9 Aflatoxins in milk, 102–103 blackberry, 9 Agaricus blazei, 151 Brazilian palm fruits, 7 Amperometric biosensor, 169 Cabernet Sauvignon pomace, 9 Anabolic abuse Caryocar brasiliense, 8 European Council Directive 23/96/EC, 110 citrus, 9 metabolomics, 111 Eugenia dysenterica, 9 proteomics, 111 Eugenia stipitata, 8 quantitative RT-PCR, 112 Eugenia uniflora, 7 strategies to trace, 110–111 Euterpe edulis, 7 survey of, 110 Euterpe oleracea, 6–7 transcriptomic technologies Gaylussacia brasiliensis, 8 DNA microarray, 120–122 Malpighia emarginata, 6 Myrciaria cauliflora, 7 Myrciaria dubia, 6 G.P.P. Lima and F. Vianello (eds.), Food Quality, Safety and Technology, 189 DOI 10.1007/978-3-7091-1640-1, © Springer-Verlag Wien 2013

190 Index Pouteria macrophylla, 7 antibody-immobilized microcantilever Rheedia brasiliensis, 8 resonator system, 171 southern Brazil, 9 Syagrus coronate, 8 bioluminescence, 171 medicinal plants choline oxidase biosensor, 170 Anacardium occidentale, 11 colorimetric response, 170–171 Anadenanthera peregrina, 10–11 electrochemical immunoassay, 172 Brazilian spirits, 11 electrochemical immunosensor, 173 Byrsonima japurensis, 10 nucleic acid aptamers, 172 Inga edulis, 11 palitoxin detection, 171 tea and seasonings, 11–12 surface plasmon resonance (SPR)-based natural defenses, 4–5 natural, for food packaging DNA biosensor, 172 additives, 182–185 toxigenic Penicillium sclerotigenum antioxidant activity, 180 carotenoids, 181 detection, 172–173 flavonoids, 181 common transducers used in, 165, 166 polyphenols, 181 components, 165–166 sources, 182 electrochemical biosensors, 165 vs. synthetic antioxidants, 180–181 pesticide, 173–175 polyphenols, 5 schematic drawing of, 165 in vegetables for small molecule determination free radicals, 22–23 polyphenols, 23–24 amperometric biosensor, 169 reduction of oxidative beta-lactam residue detection, 169 electrochemical biosensor, 167–168 phenomena, 22 limitations, 169–170 vitamin C, 23 multi-enzymatic biosensor, 168 Arac¸a´-boi fruit, 8 Biotransformation enzymes, 115–116 Ascomycetes. See Filamentous fungi Blackberry, 9 Authentication, cheese, 131–133 Brazil antioxidants, plant species B biodiversity, 4 Baccharis trimera, 11 carotenoids, 5 Bacteria toxins detection definition, 4 diversity of, 5 antibody-immobilized microcantilever fruits, 6–9 resonator system, 171 Gingko biloba, 5 medicinal plants, 10–12 bioluminescence, 171 polyphenols, 5 choline oxidase biosensor, 170 export potential, 84–85 colorimetric response, 170–171 fruit production electrochemical immunoassay, 172 drawbacks, 79–80 electrochemical immunosensor, 173 economic exploration, 78–79 nucleic acid aptamers, 172 estimates of, 77–78 palitoxin detection, 171 exports, 79 surface plasmon resonance (SPR)-based soil and climatic conditions, 78 postharvest of tropical fruits DNA biosensor, 172 handling and postharvest toxigenic Penicillium sclerotigenum preservation, 81–84 detection, 172–173 ideal harvest time, 81 Bioavailability, lycopene. See Lycopene, physiological aspects, 80–81 Brazilian palm fruits, 7 bioavailability Brazilian spirits, 11 Bioluminescence sensor, 171 Browning, 37 Biosensors Byrsonima crassifolia, 6–11 Byrsonima japurensis, 10 for bacteria and bacteria toxins detection

Index 191 C Conjugated linoleic acids (CLA), 95–96 Cabernet Sauvignon pomace, 9 Consumer protection, 130 Cachac¸as. See Brazilian spirits Cytokines, 118–119 Calcium-dependent proteolytic D system, 57–58 Dairy products. See also Cheese Calcium salts, 47 Calpain–calpastatin. See Calcium-dependent authenticity, 131–132 geographical area of cheese production proteolytic system Camellia sinensis, 11–12 altitude, 135–136 Camu-camu. See Myrciaria dubia latitude, 136–140 Cancer molds and yeast control, 140–143 PDO mark, 130–131 bacupari fruits for, 8 ripening period, 132–135 lycopene, 70–71 traditional cheese-making, 130–131 Carcasses, 54 Digestive problems, Baccharis trimera for, 11 Carotenoids, 5 1,1-diphenyl-2-picryl-hydrazyl (DPPH) Carqueja. See Baccharis trimera Caryocar brasiliense, 8 method, 5–11 Cashew, 6, 11 DNA microarray Castanea, 11 Cellulases, 152–153 DEX treatment, 120–121 Cheese gene expression, 121 altitude of cheese production, 135–136 probe-target hybridization, 120 authentication, 131–133 significance analyses of microarray (SAM) consumer protection, 130 hygiene in cheese ripening rooms test, 121 Drug metabolizing enzymes fungal proliferation, 141 mold genera, 140–141 (DMEs), 115–116 ozone treatment, 141–143 latitude of cheese production E isotopic abundance of milk, 138–139 Electrical impedance spectroscopy (EIS), 173 isotopic ratios, 137–138 Electrochemical acetylcholinesterase (AChE) PCA model, 140 percentage abundance of stable biosensor, 173–174 Electrochemical biosensors, 165, 167–168 isotopes, 137 Electrochemical immunosensor, 173 ratio in meteoric water, 137, 138 Electrochemiluminescence-based sensor, 171 sampling sites of milk, 138, 139 Electrolyzed water (EW), 47 ripening period Eugenia dysenterica, 9 Asiago cheese, 132–133 Eugenia stipitata, 8 categories, 132–133 Euterpe edulis, 7 maturation index, 133 Euterpe oleracea, 6–7 NIR technique, 133, 134 raw mean spectra, 133, 1343–135 F traditional cheese-making, 130–131 Fatty acids volatile compounds, 99–101 Chitinases, 155 animal fat, 92 Chlorine dioxide (ClO2), 47 chronic diseases, 92–93 Choline oxidase biosensor, 170 conjugated linoleic acids, 95–96 Cinnamomum, 12 in human diet, 93 Citrus, 9 of milk and cheese, 94 CLA. See Conjugated linoleic acids (CLA) omega 3 fatty acids, 93 ClO2. See Chlorine dioxide (ClO2) Fertilization, 29 Colorimetry, 170–171 Filamentous fungi, 151

192 Index Flammulina velutipes, 151 unit processing operations Flavonoids, 24 dewatering, 44 Folin-Ciocalteu reagent. See 1,1-diphenyl- edible coatings, 44 illustration, 43 2-picryl-hydrazyl (DPPH) method MAP, 45 Food of animal origin objective, 41 peeling and cutting, 43–44 aflatoxins in milk, 102–103 sanitation, 42–43 quality of time-temperature conditions, 45 washing, 43–44 concept, 92 fatty acids profile, 92–96 wounding, 36–37 selenium, 96–98 Hurdle technology, 48–49 traceability Hymenaea courbaril, 11 definition, 98 terpenes, 99, 100 I volatile compounds of milk and cheese, Intense light pulses (ILP), 48 Irrigation, 29 99–101 Fresh-cut horticultural products. See Horticultural products, fresh-cut G J Ganoderma lucidum, 151 Jabuticaba. See Myrciaria cauliflora Gastrointestinal disease, Saratudo for, 10 Gaylussacia brasiliensis, 8 L Gene profiling. See Transcriptomic Lactococcus lactis detection, 171 Lentinula edodes, 151 technologies Licuri fruits, 8 Genitourinary disease, Saratudo for, 10 Lipases, 155–156 Green tea. See Camellia sinensis Lipoxygenase (LOX) activity, 37–38 Growth promoters (GPs). See also Anabolic Lycopene abuse absorption in body, 65 effects in beef cattle, 113–115 antioxidant property of, 66 mRNA expression, 119 bioavailability on steroidogenic enzyme gene factors influencing, 67 expression, 118 isomeric forms, 68 isomerization, 68–69 H quantitative analysis method, 67 Hancornia speciosa, 8 in tomatoes, 67 Harvest, 29–30 characteristics, 65–66 Horticultural products, fresh-cut chemical structure, 64, 65 cis and trans isomers of, 64, 65 browning, 37 content in fruits and vegetables, 64 factors affecting quality and safety description, 65 and diseases, 70–71 contamination and diagnosis, 40–41 excretion of, 65 packaging, 39–40 in food sources, 69–70 preharvest factors, 38–39 high performance liquid chromatography processing operations, 39 temperature management, 40 (HPLC), 70, 71 lipoxygenase (LOX) activity, 37–38 in human body, 66 MAP, 36 preventative role in diseases minimal processing techniques, 36 technologies for quality and safety anti-inflammatory effects, 72 antimicrobial solutions, 46–47 cancers, 70–71 disinfection, 45–46 cardiovascular disease, 71 nonconventional packaging, 48–49 DNA damage, 72 UV-C radiation and intense light pulses, 47–48

Index 193 high density lipoprotein cholesterol, 72 influencing factors, 27 hypertension, 71–72 nitrogen fertilization, 26–27 toxicity of, 66 reduction, 25 sources of, 25 M toxicology, 25 Meat marbling, 55–56 Non-flavonoids, 24 Meat quality assessment, tropical Nuclear receptors (NRs), 116–117 Nucleic acid aptamers, 172 Brazilian meat market, 54–55 of Nellore cattle, 56–57 O quality characteristics, 55–56 Ocotea pretiosa, 11 tenderness Omega 3 fatty acids, 93 Organic acids, 47 calcium-dependent proteolytic system, Ozone (O3) 57–58 description, 47 crossbreed with Aberdeen Angus, 58–59 treatment, cheese ripening, 141–143 genetic factors, 60–61 shear force, 59 P slaughtering age, 60 Palitoxin (PlTX) detection, 171 Zebu cattle, 58 Pecorino cheese, 131 1-Methylcyclopropene (1-MCP), 82–84 Penicillium sclerotigenum detection, 172–173 Mezzano cheese, 132 Pequi pulp, 8 MFI. See Myofibrillar index fragmentation Peroxyacetic acid (CH3COOOH), 46 Pesticide biosensors, 173–175 (MFI) Phenolic acids. See Non-flavonoids Milk, volatile compounds, 99–100 Phenolic compounds. See Polyphenols Minimally processed products, 84–85 Phytases, 156–157 Modified atmosphere/controlled Pigments, 24 Polyphenols atmosphere, 82 Modified atmosphere packaging (MAP), description, 5 in fruits, 6–9 39–40, 45 in medicinal plants, 10, 11 Morus nigra. See Blackberry natural antioxidants, 181 Multi-enzymatic biosensor, 168 in vegetables and berries, 23–24 Murici. See Byrsonima crassifolia Polysaccharides, 151 Mycotoxin detection, 171 Post-harvest Myofibrillar index fragmentation handling, 81–82 preservation techniques (MFI), 54–55 Myrciaria cauliflora, 7 1-Methylcyclopropene Myrciaria dubia, 6 (1-MCP), 82–84 N modified atmosphere/controlled Natural antioxidant additives, 183–184 atmosphere, 82 Nellore cattle refrigeration, 82, 83 meat quality of, 56–57 quality, 29–30 meat tenderness in Pouteria macrophylla, 7 Preservation techniques calcium-dependent proteolytic system, 1-Methylcyclopropene (1-MCP), 82–84 57–58 modified atmosphere/controlled crossbreed with Aberdeen Angus, 58–59 atmosphere, 82 genetic factors, 60–61 refrigeration, 82, 83 shear force, 59 Proteases, 154–155 slaughtering age, 60 Zebu cattle, 58 Nitrate (NO3) effects of temperature, 26

194 Index Protected designation of origin (PDO), 130 polysaccharides, 151 Protected geographical indication (PGI), 130 procedure, 150 Proteolytic enzymes, 57, 154 description, 148 Pterogyne, 11 enzyme production cellulases, 152–153 Q chitinases, 155 Quality lipases, 155–156 phytases, 156–157 definition, 18 proteases, 154–155 as a function of process variants xylanases, 153–154 SSF. See Solid state fermentation (SSF) climate, 28–29 Staphylococcal enterotoxin A (SEA) fertilization and irrigation, 29 genetic aspects, 28 detection, 173 harvest and post-harvest, 29–30 Steroidogenic enzymes, 117–118 supply chain, 19–20 Stravecchio cheese, 132 of vegetables Streptomyces, 149–151 antioxidant function, 21–24 Streptomyces corchorusii, 150 nitrates, 24–27 Supply chain quality, 19–20 pigments, 24 Surface plasmon resonance (SPR)-based DNA structural and functional quality, 20–21 Quantitative real-time polymerase chain biosensors, 172 Syagrus coronate. See Licuri fruits reaction (qRT-PCR) Syagrus romanzoffiana, 7 biotransformation enzymes, 115–116 Synthetic antioxidants, 180–182 cytokines, 118–119 gene panel, 119–120 T nuclear receptors, 116–117 Terpenes, 99, 100 steroidogenic enzymes, 117–118 Toxicity R of lycopene, 66 Reactive oxygen species (ROS), 4 of nitrates, 25 Refrigeration, 82, 83 Traceability Rheedia brasiliensis, 8 definition, 98 RNA sequencing (RNA-Seq), 122 terpenes, 99, 100 ROS. See Reactive oxygen species (ROS) volatile compounds of milk and cheese, Rubus. See Blackberry 99–101 S Transcriptomic technologies Sanitizers, 43 Saratudo. See Byrsonima japurensis definition, 112 Selenised yeast, 97 DNA microarray, 120–122 Selenium (Se), 96–98 effect of GPs, 113–115 Small molecule determination quantitative real-time RT-PCR, 112–120 RNA-Seq, 122 amperometric biosensor, 169 Tropical fruits beta-lactam residue detection, 169 export potential, 84–85 electrochemical biosensor, 167–168 postharvest of limitations, 169–170 multi-enzymatic biosensor, 168 handling and postharvest Solid state fermentation (SSF) preservation, 81–84 actinomyces, 148–149 bioactive substances, production of ideal harvest time, 81 physiological aspects, 80–81 carotenoids, 151 production classification, 149–151 drawbacks, 79–80 economic exploration, 78–79 estimates of, 77–78 exports, 79 soil and climatic conditions, 78

Index 195 U W UV-C radiation, 47–48 Whole transcriptome shotgun sequencing V (WTSS), 122 Vecchio cheese, 132 Wounding, 36–37 Vitamin C, 6 X Xylanases, 153–154